The eukaryotic reverse transcriptase telomerase elongates chromosome ends by addition of single- stranded telomeric repeats. This new DNA synthesis is required to balance the loss of DNA inherent in the incomplete replication of chromosome ends by conventional DNA polymerases. The long-term objective of research funded by this RO1 is to elucidate the mechanisms that underlie the unique catalytic activity and biological function of telomerase. Understanding telomerase mechanism has direct relevance for the improvement of human health. Human gene mutations that reduce telomerase activity cause proliferative deficiencies, while aberrantly high telomerase activation gives cancer cells their lethal capacity for indefinite proliferation. Research in the next funding period will continue to provide fundamental gains in knowledge about telomerase structure and mechanism, new insights in the developing paradigm of how RNA motifs specialize the function of a ribonucleoprotein enzyme, new principles for sequence-specific handling of single-stranded DNA, and groundwork for the design of clinically useful strategies of telomerase activation and inhibition. Telomerase evolved an elaborate catalytic cycle to specialize its role in telomere maintenance, requiring the collaboration of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and other subunits of a biologically active telomerase holoenzyme. The Specific Aims make particular use of knowledge gained about Tetrahymena telomerase in the current funding period. Aim 1 experiments will establish mechanisms for TER motif function in the catalytic cycle using RNA-RNA and RNA- protein interaction assays and activity assays sensitized to detect specific steps of catalytic cycle progression. Insights about mechanism will be tested for conservation in human telomerase. Results from Aim 1 will expand knowledge about telomerase and also extend a general paradigm for ribonucleoprotein functional specialization. Aim 2 experiments will address how telomerase recognizes single-stranded telomeric-repeat DNA, exploiting our recent reconstitution of the first complete telomerase holoenzyme in vitro. Guided by atomic-resolution structures of telomerase holoenzyme DNA binding domains, we will use activity assays and assays of protein and DNA interaction to address how telomerase dynamically handles single-stranded telomeric repeats. Aim 3 experiments will exploit this knowledge to establish the requirements for telomerase-telomere interaction in vivo, using transgene integration and gene replacement methods along with affinity purification and chromatin association assays. Together Aims 2 and 3 will reveal new principles of protein-DNA interaction and elucidate the determinants of telomerase specificity for telomeres. PUBLIC HEALTH RELEVANCE: The proposed studies of telomerase from the enabling model system Tetrahymena and from human cells will increase our understanding of the biochemical mechanisms that give this enzyme its unique catalytic activity and biological function. Insights about telomerase mechanism will provide opportunities to improve human health, because diseases of insufficient telomerase activation including some cases of inherited bone marrow failure, aplastic anemia, and pulmonary fibrosis could be treated therapeutically by telomerase activation. Also, because human cancers require aberrantly high telomerase activation for continued growth, telomerase inhibitors have widely recognized promise as selective anti-tumor agents.